Because of their unique self-renewal and pluripotent properties, human embryoninc stem (hES) cells hold great promise for regenerative medicine. However, the molecular mechanisms controlling hES cell self- renewal and pluripotency remain largely unknown. Mammalian ES cells rely on a complex network of transcriptional regulators and epigenetic modulators such as Nanog, Oct4, and Polycomb to maintain gene transcription programs necessary for self-renewal and pluripotency. How these factors determine epigenetic marks and ES cell fate remains one of the most important questions. To this end, we undertook a proteomic approach to isolate and characterize protein complexes from mouse ES cells. Proteins in the endogenous Nanog complex have been identified by affinity purification and mass spectrometry. Interestingly, we found that the Nanog complex contains multiple transcriptional repressers, including components of the Polycomb PRC1 complex. Polycomb proteins mediate epigenetic modification of histone chromatin and transcription silencing, and have been implicated in cell fate determination and developmental control. These findings have led to the hypothesis that Nanog recruits Polycomb transcription repression complexes to regulate epigenetic marks on histones and hence ES cell self-renewal and differentiation. Little is known about the human Nanog complex; however, we expect that it exhibits both similarities and differences relative to the mouse Nanog complex. Here we propose to biochemically and genetically analyze the Nanog protein complex and Nanog-Polycomb interactions in hES cells through the following specific aims: Specific Aim 1. To determine the structural organization of the Nanog complex and Nanog-Polycomb interactions in human ES cells. Specific Aim 2. To investigate the molecular mechanisms by which the Polycomb PRC1 complex and epigenetic modifications regulate Nanog-mediated gene expression. Specific Aim 3. To determine the roles of Nanog complex and Nanog-Polycomb interaction in hES self-renewal and differentiation. This project interacts extensively with Projects 2 and 3, 4, as we will investigate whether the proteins under study (GCNF, p53, and THAP11) are found to interact with Nanog or Nanog-complex proteins. We will also compare the genes bound by NANOG with those bound by these proteins in ChlP-on- chip studies. We will also examine whether perturbation of components of the PRC1 complex leads to changes in regulation of p53, GCNF, and THAP11 through changes in histone modifications near these genes. These studies will lead to a detailed view on how these important proteins interact to regulate hES pluripotency. This project will utilize the hES Training and Culture Core to obtain large-scale cultures of hES Cells for proteomic studies, as well as smaller cultures for routine experiments. Several genetically modified hES cells lines will be constructed by the Genetic Modification Core.